Class switch recombination (CSR) is the somatic DNA rearrangement process by which B lymphocytes change their heavy chains from IgM to IgG, IgA or IgE. This DNA recombination process is distinctive among all eukaryotic (and prokaryotic) recombination mechanisms because it is the only one in which the recombination occurs within long repetitive regions in a manner that is dependent on transcription through the region such that a G-rich transcript is generated. Understanding of the mechanism of this process is important for immunologic reasons (infection, allergy, autoimmunity) and for its cancer-relevance (neoplastic translocations that occur within the class switch sequences, such as the t(8;14)). In the previous funding cycle, we showed that class switch regions assume a distinctive structure within the chromosomes of appropriately stimulated B cells. This structure is an R-loop in which the G-rich (nontemplate) strand of the repetitive switch regions is single-stranded for lengths exceeding 1 kb. The G-rich RNA that is generated by transcription through the switch region remains annealed to the C-rich (template) strand of the switch region. These results explain the in vivo orientation-dependence of the transcription requirement, which we documented in our earlier switch recombination work. In the current proposal, Aim 1 examines whether the chromosomal R-loop is limited to the switch repeats and whether it is affected by splicing. Aim 2 examines the mechanism of R-loop formation, and this is optimally done in vitro where we probe the structure with enzymes; study the repeat length and sequences needed for R-loop formation; vary the rate of RNA polymerase movement; and vary the number of hybrogen bonds in the R-loop. Aim 3 examines the interface between AID enzyme activity and R-loops. Can purified AID deaminate both DNA strands in the R-loop? Is the proximity of WRC sequence motifs on the two DNA strands critical for double-strand break formation? Aim 4 tests whether DNA ligase IV is required for CSR. This aim is directed at providing some clarification of what end joining pathway is used for class switch recombination. Hence, this proposal tests in vivo substrate structure, its mechanism of formation, the action of AID enzyme on that structure, and the pathway for rejoining the ends after breakage has occurred.